The broad and long-term goal of this project is to understand the patterning of coronary arteries and their underlying mechanisms. The coronary circulation starts when the passive diffusion of oxygen and nutrients is no longer sufficient for the heart growth and function. The coronary arteries must connect to the aorta through the coronary ostia, the openings at the aortic sinus, to receive the oxygenated blood and nutrients from systemic circulation. We have generated new genetic mouse models to study the developmental and molecular mechanisms of the coronary-aorta connection and coronary patterning. Preliminary results have shown that the myocardial vascular endothelial growth factor-a (Vegfa) is necessary for the connection to form. We have also found that the main receptor for Vegfa, Vegfr2, as well as transcription factor Nfatc1 are co- expressed in the aortic sinus endothelium. Additionally, the aortic sinus is surrounding by hypoxic cardiomyocytes that produce a high level of Vegfa. Building upon these findings, this project is to study in mice when and where the myocardial Vegfa is required for the coronary-aorta connection and whether its function is hypoxia-dependent. We will also address whether Vegfr2 and Nfatc1 function together to regulate the connection process. Specifically, we will genetically disrupt Vegfa, Vegfr2, or Nfatc1 in a spatiotemporal manner during late coronary artery development. Ablation of the hypoxic cardiomyocytes or their expression of Vegfa will establish the role of hypoxia in coronary artery patterning and ostium formation. Deletion of Vegfr2 and Nfatc1 in the endocardium of the aortic sinus will ascertain their roles and potential interactions in these processes. Morphologic, cellular, and molecular analysis of the developing coronary arteries and ostia in these mouse mutants will identify previously unknown developmental mechanisms and molecular interactions involved in late coronary artery development. The information will help understand the etiology of congenital coronary anomalies.